Paediatrics (RCH) - Theses
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ItemInvestigating the biology of paediatric T cell acute lymphoblastic leukaemia to facilitate more effective individualised therapy( 2019)Acute lymphoblastic leukaemia (ALL) takes up the highest percentage of paediatric cancer. The treatment requires intensive chemotherapy for two to three years, as well as haematopoietic stem cell transplantation for poor-prognosis cases. Compared to B cell lineage ALL (B-ALL), T cell lineage ALL (T-ALL) has a lower 5-year event free rate, higher rate of relapse, and a worse outcome for relapsed cases. Individualised therapy, targeting at oncogenic changes in each patient, can make treatment more effective and less harmful. This requires understanding of the oncogenic biology of each individual leukaemia. We have attempted to develop a T-ALL model based on hiPSC-derived T cells, which will be in human origin, maintain normal genetic pattern, mimic in vivo T cell development, and can be massively produced for high throughput lab work. This model may make up for the shortcomings of conventional leukaemia cell lines and mouse models. This project investigates the biology of T-ALL by focusing on two novel fusion genes – TCF7-CSF1R and ETV6-CRX – identified by RNA sequencing of paediatric T-ALL patient samples. We have shown that TCF7-CSF1R is sufficient to immortalise mIL-3 dependent Ba/F3 cells. The ETV6-CRX fusion gene is anticipated to block differentiation. Establishing consistent expression of this fusion will require further optimization. The feasibility of setting up a hiPSC-derived T-ALL model was also assessed, with respect to protein expression in human T-ALL/lymphoma cell lines, hiPSC differentiation efficiency, hiPSC-derived T cell lentiviral infection rate, and cytokine withdrawal during differentiation. This project provides potential directions for improvement of methods for exogenous gene expression, such as the usage of CRISPR-Cas9 based techniques to introduce gene modifications for fusion genes such as ETV6-CRX that are difficult to express, particularly in hiPSC-derived T cells that have a low viral infection level. The T cell differentiation protocols also need to be optimised to make the T cell production easier and efficient. Detailed functional assay during T cell differentiation needs to be conducted in the future. In this thesis, Chapter 1 presents the background of this project; a literature review introducing human haematopoietic system, in vivo thymocyte development, paediatric TALL, novel oncogenic fusion-related genes, and in vitro T cell generation; the aims and hypothesis. Chapter 2 introduces the methods and materials used in this project. Chapter 3 presents the identification, cloning, and expression of novel fusion genes. Chapter 4 investigates the ability of these novel fusion genes to support cell survival and proliferation in conventional Ba/F3 cell line. Chapter 5 assesses the feasibility of setting up the hiPSC-derived T-ALL model. Chapter 6 makes a discussion on the results and concludes the whole project.